1. Field of the Invention
The present invention relates to a method and apparatus for conducting agglutination immunoassay by containing, in a container, a test sample suspected of containing a desired analyte and a reagent which comprises magnetic-material containing particles, which have been sensitized to permit specific binding to the desired analyte.
2. Discussion of Background
Conventionally, agglutination immunoassay has been conducted by utilizing agglutination reaction which is caused to occur by antigen-antibody reaction to detect the presence or absence of the desired analyte such as an antibody or antigen in the test sample.
It is possible to detect whether or not such an agglutination reaction occurs by allowing the mixture of the test sample and the reagent to stand stationarily for a while and to then inspect the state or shape of an agglutination pattern composed of the sensitized particles on which the antigen or antibody is bound or immobilized, or the state or shape of unbound reagent.
Such conventional immunoassay, however, is not quick and the mixture of the test sample and the reagent must be maintained for some time under conditions completely free from vibrations.
Under such circumstances, the Applicants of the present invention have proposed an agglutination immunoassay in European Patent 426170. In the agglutination immunoassay, the test sample suspected of containing the desired analyte is contacted with a reagent comprising magnetic-material containing particles, which have been sensitized to permit specific binding to the desired analyte, in a container such as wells of microtiter plate.
In the agglutination immunoassay, the analyte is bound or immobilized on the sensitized particle. The magnetic-material containing particles, which have bound to the desired analyte, are magnetically precipitated to the bottom of the container by application of magnetic force.
After precipitation, the container in which the contact occurs is allowed to stand at an inclination.
On inclination, the precipitated particles will flow under the effect of gravity if no immune binding reaction has occurred. The absence of flow indicates that the desired analyte is present and bound to the sensitized particles. Thus, by the presence or absence of such a flowed developed pattern (hereinafter referred to as a developed pattern), the presence or absence of the agglutination reaction, and accordingly the presence or absence of the desired analyte is judged. This assay is quick and reliable for a large number of test samples.
In order to further speed up this assay, and also to make this assay more reliable without depending upon operators' individual skill, the Applicants of the present invention have further proposed in European Application 625708A a high speed automated apparatus for the assay by use of an optical sensor which is capable of detecting the shape of the developed pattern of the precipitated particles at the bottom of each well of microtiter plate.
More specifically, in the apparatus, there is provided a line optical sensor comprising micro optical sensors which are arranged side by side in the extending direction of the line optical sensor, in the same number as that of the wells in each column of the microtiter plate, in such a manner that each micro optical sensor can measure the length of a crossing portion of a line that crosses the developed pattern in a predetermined direction in the corresponding well.
In order to detect the shape of the developed pattern accurately, the line optical sensor is moved to the right and left for the sample in each well so that the measurement of the length of the crossing portion of the line is carried out at three representative points in total.
In this method, the above-mentioned measurement is carried out for each developed pattern. However, unless the positioning of the measurement for the three lines is carried out appropriately and accurately for each developed pattern, the shape of the developed pattern cannot be detected accurately. Therefore, it is required that the positioning of the measurement for the three lines be made appropriately and accurately. Furthermore, it is required that the number of the lines to be measured be maximized.
However, the greater the number of the lines for the measurement of the length, the longer the time required for the assay; and more accurate operating means is required for handling the microtliter plates and the optical sensor in order to perform accurate and appropriate positioning of the measurement of the length of the three lines.
The Applicants of the present invention have further proposed in Japanese Laid-Open Patent Application 5-297001 an apparatus for the above-mentioned immunoassay, which utilizes a CCD camera for detecting the shape of the previously mentioned developed pattern in agglutination immunoassay.
More specifically, in the above apparatus, image signals are obtained by the CCD camera from the microtiter in its entirety and processed so as to calculate the length of each developed pattern from the changes in luminance detected from the obtained image signals.
The values obtained by this apparatus, however, so scatter that reliable detection data cannot always be provided with respect to the shape of the developed pattern.